Detergents comprising cellulases

ABSTRACT

Laundry detergent compositions containing one or more cellulases having a ratio of tensile strength loss to antipilling properties of less than 1. The cellulases may be obtained from CBS 9.93 or CBS 670.93.

[0001] The present invention relates to the use of novel cellulases withimproved properties in detergents and aqueous laundry solutions. Theinvention further relates to detergents and detergent additivescomprising the novel cellulase.

[0002] Cellulases, also called cellulolytic enzymes, are enzymes whichare capable of the hydrolysis of the β-D-glucosidic linkages incelluloses. Cellulolytic enzymes have been divided traditionally intothree classes: endoglucanases, exoglucanases or cellobio-hydrolases andβ-glucosidases (Knowles, J. et al. (1987), TIBTECH 5, 255-261)Cellulolytic enzymes can be produced by a large number of bacteria,yeasts and fungi. Microorganisms that produce cellulases are describedin for example GB-A-2094826.

[0003] Several applications have been developed for the use ofcellulolytic enzymes:

[0004] degrading (wood)cellulose pulp into sugars for (bio)ethanolproduction;

[0005] several textile treatments like ‘stone washing’ and‘biopolishing’;

[0006] application in detergent compositions.

[0007] The use of cellulases in detergent compositions started withcellulases capable of reducing the harshness, i.e softening, of cottoncontaining fabrics, as described in for example GB-B-1358599.

[0008] It is further known that detergent compositions comprisingcellulases are effective in removing dirt, i.e. cleaning. The efficiencyof cellulolytic enzymes, cellulases, in terms of cleaning textile hasbeen recognized for some time. GB-A-2075028, GB-A-2095275 andGB-A-2094826 disclose detergent compositions with cellulose for improvedcleaning performance.

[0009] It is also known in the art that cellulases can act as a colorclarifying agent in laundry detergents. After repeated washing of soiledfabrics, cotton containing fabrics appear to be greyish, most probablydue to disrupted fibers caused by mechanical action. The fibers are tomup resulting in disordered fibers which are broken. The use ofcellulases as color clarification agents for colored fabrics has beendescribed in EP-A-0220016. Actually cellulase mixtures from the fungalstrain Humicola insolens (DSM 1800) are commonly used in detergents toresult in antipilling and color revival properties. The cellulolyticenzyme system produced by the wild type microorganism is available underthe trade name of Celluzyme® by Novo-Nordisk. In addition a cloned(single) cellulase from the same origin under the trade name Carezyme®is also used in detergents.

[0010] The main disadvantage of the cellulases known in the art showingcolor clarification is that these enzymes agressively degrade thecellulose containing fabrics which results in damage by undesirable lossof tensile strength of the fabrics.

[0011] On the other hand cellulases known in the art showing goodcleaning properties show hardly any color clarification effects. Thefirst commercial detergent with cellulases in the world contained abacterial cellulase. This enzyme represents an above mentioned alkalineendoglucanase from a Bacillus species that does not attack cellulosefibers. The enzyme is described to give a cleaning effect duringwashing. No effects with respect to anti-pilling or color revival havebeen described for this enzyme.

[0012] From the above it will become clear that it is still desirable toprovide for improved cellulases in detergent applications. Usingmixtures of cellulases, as suggested in international patent applicationWO-A95102675, is supposed to provide the above mentioned performances inlaundry washing, but to our knowledge it has not previously beenpossible to use single enzymes providing all these characteristics whenapplied in laundry washing.

[0013] Surprisingly it has been found that the use of certain singlecellulases which are capable of cleaning, antiredeposition, colorclarification and antipilling performance in laundry washing does not atall result in unacceptable damage to the textiles washed.

[0014] Accordingly, the present invention relates to the use of a singlecellulase with a ratio of tensile strength loss (TSL, as hereindefinded) to antipilling properties (AP, as herein defined) below 1 inaqueous laundry solutions.

[0015] To measure tensile strength loss is a way to measure damagecaused by mechanical stress or enzymatical action on fibers. It is to beunderstood that for the purpose of the present invention cotton fiberhas to be used. The method measures the tensile strength of singlefibers under wet conditions. It is described in the German Standard DIN53, 857, part 1, as well as in the International Standard ISO 2267.

[0016] As the effect normally shows up in a significant amount onlyafter about 20 to 25 wash cycles, there is always some tensile strengthloss due to the mechanical forces acting on the cotton fiber during thewashing process. Therefore the tensile strength loss of a control fabricwashed without cellulases using the same formulation of detergent andthe same type of washing machine and washing programme has to besubtracted. To calibrate the values, a preparation of the (single)endoglucanase V from Humicola insolens (EG V) in equal amounts ofenzymatic protein in the detergent is used as a standard and the valueof the tensile strength loss for this sample minus the control value ofdetergent without cellulase is taken as a TSL of 100%. This cellulase EGV has been described for example in the international patent applicationWO 91/17243. The amount of protein can be measured for example by usingthe BCA Pierce method as described by R. E. Brown et al. in Anal.Biochem. 1989, vol. 180, p. 136 - 139.

[0017] A preparation of an above mentioned Bacillus cellulase availablefrom Kao Corp. under the trade mark KAC® 500 or KAC® 700 may be used ascomparison, resulting in general in a very low tensile strength loss ascompared to the control washing experiment with no cellulase present.

[0018] The attack of cellulases on protruding microfibrils, pills andcotton fluff on the surface of a cotton fabric results in an opticallyvisible removal of that pills. To test the effect, washings are to beperformed using a detergent with and without cellulase. as described forthe detremination of TSL. The antipilling effect, too, can best be seenafter an increasing number of wash cycles. Therefore a number of 15 to40 wash cycles are generally used to demonstrate this effect ofcellulases.

[0019] There are three different methods that can be used forquantification of this effect:

[0020] 1. visual evaluation by a test group (panel)

[0021] 2. measurement of light reflection (L-value of the CIELAB-system)

[0022] 3. determination of the cotton fluffs by means of opticalmeasurement

[0023] The determination using the L-value of the CIELAB-system[Commission Internationale de I'Éclairage] was described by U. Hotz inTenside Surf. Det. 1993, vol. 30, page 388.

[0024] The optical measurement system, which is used in the preferredmethod of determining the antpilling properties, usually consists of alight source, a microscope tube and a CCD color camera recording thelight reflected from the surface of a fabric. Depending on the amount ofpills and fluff on the surface of the fabric the amount of reflectedlight as measured by digital image analysis changes. Such a system canbe used to measure quantitatively the amount of pill and fluff onfabrics, normally after 15 to 40 wash cycles depending on the type andactivity of the cellulose added to the detergent. An optical systemwhich can be used to measure the degree of pilling has been described byT. Müller-Kirschbaum and H. Grundmann in SÖFW, vol. 118 (1992), p.483-499.

[0025] Whatever method is used to determine the antipilling effect ofthe cellulase to be tested, the standard cellulase EG V has to be testedunder the same conditions and it's effect has to be determined by thesame method, taking into account the value resulting from the use of thedetergent without cellulase. The value obtained for EG V is taken asAP=100 %.

[0026] As can be seen from this definition, the known cellulase EG Vfrom Humicola insolens has a ratio of TSL to AP of 1. As the abovementioned Bacillus cellulase available from Kao Corp. under the trademark KAC® 500 or KAC® 700 has a low AP and a very low TSL, it can beseen that also the ratio for this cellulase is approximately 1.Cellulases which may be used according to the invention, especially indetergents, have a ratio of TSL to AP as much as possible below 1,preferably below 0.8 and more particularly in the range of 0.001 to 0.5.A ratio of TSL to AP of for example 0.5 means that only 50% of tensilestrength loss is seen at an enzyme concentration yielding the sameantipilling effect as the standard cellulase.

[0027] The aqueous laundry solution preferably comprises cellulaseaccording to the definition given above in concentrations of 0.01 mg/lto 02 mg/l, more particularly 0.015 mg/l to 0.1 mg/l. Theseconcentrations refer to the weight of celluolytic protein. In additionall ingredients normally found in laundry solutions can be present

[0028] Another aspect of the present invention is the use of a singlecellulase with a ratio of TSL to AP below 1 to provide an anti-greyingeffect to fabrics, especially colored fabrics.

[0029] In another aspect of the invention a single cellulase with aratio of TSL to AP below 1 is used to provide a softening effect tofabrics.

[0030] The present invention also relates to the use of a singlecellulase with a ratio of TSL to AP below 1 to provide colorclarification or to inhibit color deterioration of fabrics, especiallycolored fabrics.

[0031] The present invention further relates to the use of a singlecellulase with a ratio of TSL to AP below 1 to inhibit the wrinkling offabrics and to ease the ironing of fabrics.

[0032] We found that the use of a single cellulase according to thedefinition of the invention, unlike previously known mixtures ofcellulases which provide color clarification, does not degrade cotton toan undesirable level causing tensile strength loss.

[0033] It Is further found that in using a cellulase of the definitionaccording to the invention, unlike previously known cellulases whichprovide color clarification, the enzyme does not accumulate on thefabric after repeated laundry washing.

[0034] In another aspect, the invention is directed to detergentcompositions, detergent additives and fabric softener compositionscomprising a single cellulase according to the definition given above.

[0035] As noted before, the present invention generally relates to theuse of novel cellulases. However, prior to disclosing this invention inmore detail, the following terms will be defined:

[0036] “Cellulase” is a generic name for enzymes acting on cellulose andits derivatives, and hydrolysing them into glucose, cellobiose orcellooligosaccharides. The term “single” cellulase used herein isintended to mean a cellulase which is produced by one gene.

[0037] “Obtainable from” an organism in connection with a cellulasemeans that such cellulase has an amino acid sequence which correspondsto the amino acid sequence of a cellulase which may be obtained fromthat organism.

[0038] “Derivative” is intended to indicate a protein which is derivedfrom the native protein by addition of one or more amino acids to eitheror both the C- and N-terminal end of the native protein, substitution ofone or more amino acids at one or a number of different sites in thenative amino acid sequence, deletion of one or more amino acids ateither or both ends of the native protein or at one or more sites in theamino acid sequence, or insertion of one or more amino acids at one ormore sites in the native amino acid sequence. The preparation of aderivative is usually achieved by modifying a DNA sequence which encodesfor the native protein, transformation of that DNA sequence into asuitable host and expression of the modified DNA sequence to form thederivative protein. The derivative of the invention includes peptidescomprising altered amino acid sequences in comparison with a precursorenzyme amino acid sequence (e.g., a wild type or native state enzymeaccording to the present invention) and which peptides retain acharacteristic enzyme nature of the precursor enzyme but which havealtered properties in some specific aspect. For example, an alteredcellulase may have an increased pH optimum or increased temperatureresistance but will retain its characteristic cellulase activity.Derivatives also includes chemical modifications of amino acid residueswithin the enzyme molecule.

[0039] “Host cell” means a cell which has the capacity to act as a hostand expression vehicle for a recombinant DNA vector comprising DNA whichencodes for the native protein or a derivative.

[0040] The term “cleaning” means the removal of dirt attached tolaundry.

[0041] The term “pilling” in this respect is the formation of pills andfuzz on the surface of cotton containing fabrics due to broken ordisordered fibers.

[0042] The term “antipilling” is used to describe the prevention of theformation of pills and fuzz on the surface of cotton containing fabricsas well as the removal of pills and fuzz from cotton containing fabrics.Antipilling normally results in color clarification when colored cottoncontaining fabrics are treated

[0043] The term “color clarification” in this respect is thereestablishment of the attractive fresh look of colored fabricscontaining or consisting of cellulose based fibers, which have developeda greyish appearance by treatment, especially with laundry detergents,of the colored fabric.

[0044] The term “redeposition” in this respect is deposition of dirt orcolor components that were removed from these textiles or fabrics duringa laundry washing or textile treatment.

[0045] The term “antiredeposition” in this respect is the action ofcellulase to prevent or diminish the redeposition of dirt and colorcomponents on the fabric.

[0046] By a “laundry solution” is meant an aqueous solution used forwashing, rinsing or conditioning, e.g. softening fabrics.

[0047] In a preferred aspect, the present invention relates to the useof a cellulase which is obtainable from microorganisms which aredeposited according to the Budapest Treaty on the InternationalRecognition of the Deposits of Microorganisms for the Purposes of PatentProcedures, at the Centraal Bureau voor Schimmelcultures, Baam, TheNetherlands on Dec. 23, 1993 under deposition numbers CBS 669.93 and CBS670 93 (described in international patent application WO-A-95/18219).This strains have been classified as new species of the genus Bacillus,which do not belong to any of the presently known rRNA-groups ofBacillus. As used herein, the deposited species will be referred to asCBS 669.93 and CBS 670.93.

[0048] The microorganisms may be obtained for example from water andsoil samples collected in alkaline environments such as alkaline soilsand soda lakes.

[0049] The microorganisms have subsequently been screened using acarboxymethyl cellulose (CMC)-agar diffusion assay. Strains which showeda clearing zone in this test were isolated as potential cellulaseproducing strains. Genomic gene libraries of the alkali tolerantcellulase producing strains were constructed. Recombinant clones werescreened by agar diffusion on CMC-agar. Recombinant-clones that showedclearing zones around the colony were isolated. Single cellulases wereproduced by fermentation of the recombinant clones in 4*YEP-medium for48 hours at 30° C. The obtained single cellulases, optionally purifiedas described in Example 1, were tested in the tests defined above tomeasure TSL and AP.

[0050] Surprisingly it was found that the cellulases obtainable from CBS670.93 or CBS 669.93 show a good performance in both tests and have aratio of TSL to AP below 1.

[0051] In a preferred embodiment of the invention, an approximately 50kD cellulase (calculated on the basis of the amino acid sequence (SEQ IDNo.2) of the mature protein) derived from CBS 670.93 (referred to as“BCE 103” herein) is used. It has been revealed by analyzing the geneencoding the amino acid sequence of the approximately 50 kD cellulasethat this cellulase is 89% identical in sequence and 92.5% similar insequence to the cellulase CelA of Bacillus sp. N-4 (Fukumori et al., J.Bacter., vol. 168, pp. 479-485) by using the TFastA program (SequenceAnalysis Software Package 6.0 of Genetic Computer Group. University ofWisconsin, Biotechnology Center, Madison, Wis.) as described by Pearsonand Lipman in Proc. Nat. Acad. Sci., vol. 85, pp. 2444-2448 (1988). Theamino acid sequence of BCE 113 is given in SEQ. ID. No.3. The presentinvention further encompasses the use of cellulases with an amino acidsequence which have greater than 89%, preferably greater than 95%sequence identity and/or greater than 92.5%, preferably greater than 97%sequence similarity thereto, and detergents comprising such a cellulase.

[0052] In an equally preferred embodiment of the invention, anapproximately 63 kD cellulase (calculated on the basis of amino acidsequence of the mature protein) derived from CBS 669.93 (referred toherein as “BCE 113”) is used. It has been revealed by analyzing the geneencoding the amino acid sequence of the approximately 63 kD cellulasethat this cellulase is 58% identical in sequence and 72% similar insequence to the cellulose CelB of Bacillus lautus (Jorgensen et al,Gene, vol. 93 (1990), p. 55-60) by using the TFastA program (SequenceAnalysis Software Package 6.0 of Genetic Computer Group, University ofWisconsin, Biotechnology Center, Madison, Wis.) as described by Pearsonand Uppman in Proc. Nat. Acad. Sci., vol. 85 (1990), p. 2444-2448. Theamino acid sequence of BCE 113 is given in SEQ ID No.3. The presentinvention further encompasses the use of cellulases with an amino acidsequence which have greater than 58%, preferably greater than 80% andmore particularly greater than 90% sequence identity and/or greater than72% preferably greater than 80% and more particularly greater than 90%sequence similarity thereto, and detergents comprising such a cellulose.

[0053] A cellulase which may be used in detergents according to thepresent invention in addition to having a ratio of TSL to AP below 1usually performs well in the Antiredeposition Test as described inExample 4. Whiteness maintenance of white fabric is measured by areflectance measurement. The higher the reflectance value, the moreeffective the tested cellulase is in antiredeposition performance. Theyalso perform well in the Softening Test as described in Example 4.Depilling is the removal of fibrils and/or microfibers that aredisordered and/or broken which usually make a colored cotton containingfabric look greyish. The more disordered and/or broken fibrils areremoved the better the colored cotton containing fabrics look. Depillingeffectiveness can be judged by panels or can be quantified by an imageanalysis system as specified above for the measurement of AP. Cellulaseswhich fulfil the requirement of the ratio defined above usually exhibitthe following properties: They show a delta REM of at least 4 units,preferably at least 5 units, in the Anti Redeposition Test as defined inthe Examples, and they show a depilling result which is at leastcomparable to that of the cellulase obtainable from CBS 670.93.

[0054] The cellulases which can be used according to the presentinvention may be produced by a process which can be developed usinggenetic engineering. As a first step the gene encoding the cellulase ofthe present invention can be cloned using λ-phage (expression) vectorsand E. coli host cells. Alternatively PCR cloning using consensusprimers designed on conserved domains may be used. Expression of thegene encoding the cellulase of the present invention in E. coli is shownto give an active protein.

[0055] After a first cloning step in E. coli, a cellulase gene can betransferred to a more preferred industrial expression host such asBacillus or Streptomyces species, a filamentous fungus such asAspergillus, or a yeast. High level expression and secretion obtainablein these host organisms allows accumulation of the cellulase of theinvention in the fermentation medium from which they can subsequently berecovered.

[0056] Cellulases according to the invention are preferably used inamounts of 8·10⁻⁵% by weight (0.8 ppm) to 8·10⁻³% by weight (80 ppm),more particularly 1·10⁻⁴% by weight (1 ppm) to 4·10⁻³% by weight (40ppm), referring to the cellulolytic protein, in detergents. Detergentcompositions comprising a cellulase defined according to the inventionmay additionally comprise surfactants which may be of the anionic,non-ionic, cationic, amphoteric or zwitterionic type as well as mixturesof these surfactant classes. Detergent compositions of the invention maycontain other detergent ingredients known in the art, as e.g. builders,bleaching agents, bleach activators, anti-corrosion agents, sequesteringagents, soil release polymers, perfumes, other enzymes, enzymestabilizers, etc.

[0057] Suitable builders are in particular those from the classes ofpolycarboxylic acids, more particularly polymeric acrylic acids,methacrylic acids, maleic acids, copolymers thereof and oxidizedcarbohydrates, as described in international patent applicationWO-A-93/16110, layer silicates, more particularly bentonites,alumosilicates, more particularly zeolites, crystalline or amorphousalkali metal silicates, more particularly sodium silicate, and alkalimetal carbonates, more particularly sodium carbonate. The polycarboxylicacids mentioned are normally used in the form of their alkali metalsalts, more particularly in the form of their sodium or potassium salts.The zeolites preferably incorporated are in particular those of the A, Por X type or mixtures thereof. Preferred alkali metal silicates arethose with molar ratios of SiO₂ to alkali metal oxide of 1.5 to 3.0.Builders such as these are preferably present in detergents according tothe invention in quantities of 20% by weight to 80% by weight.

[0058] Nonionic surfactants may be present in the detergents accordingto the invention, preferably in quantities of not more than 10% byweight and, more preferably, in quantities of 2% by weight to 6% byweight, based on the detergent as a whole. Suitable nonionic surfactantsare alkyl polyglycosides containing 10 to 22 carbon atoms in the alkylcomponent and alkoxylates, particularly ethoxylates and/or propoxylates,of linear or branched C₁₀₋₂₂ and preferably C₁₂₋₁₈ alcohols. The degreeof alkoxylation of the alcohols is between 1 and 20 and preferablybetween 3 and 10. They may be prepared in known manner by reaction ofthe corresponding alcohols with the corresponding alkylene oxides. Thefatty alcohol derivatives are particularly suitable, although theirbranched-chain isomers, more particularly so-called oxoalcohols, may beused for the production of useable alkoxylates. Accordingly, theethoxylates of primary alcohols containing linear dodecyl, tetradecyl,hexadecyl or octadecyl radicals and mixtures thereof are particularlyuseful. In addition, corresponding ethoxylation and/or propoxylationproducts of alkyl amines, vicinal diols and carboxylic acid amides,which correspond to the alcohols mentioned in regard to the alkylcomponent, and of alkyl phenols containing 5 to 12 carbon atoms in thealkyl component may also be used.

[0059] Suitable anionic surfactants are in particular those of thesulfate or sulfonate type, although other types, such as soaps,long-chain N-acyl sarcosinates, salts of fatty acid cyanamides or saltsof ether carboxylic acids, which may be obtained from long-chain alkylor alkyl phenyl polyglycol ethers and chloroacetic acid, may also beused. The anionic surfactants are preferably used in the form of thesodium salts. Surfactants are preferably present in quantities of 2% byweight to 30% by weight and more preferably in quantities of 5% byweight to 20% by weight.

[0060] Particularly suitable surfactants of the sulfate type are thesulfuric acid monoesters of long-chain primary alcohols of natural andsynthetic origin containing 10 to 20 carbon atoms, i.e. the sulfuricacid monoesters of fatty alcohols such as, for example, coconut oilfatty alcohols, tallow fatty alcohols, oleyl alcohol, or the C₁₀₋₂₀oxoalcohols and those of secondary alcohols of the same chain length.The sulfuric acid monoesters of aliphatic primary alcohols, secondaryalcohols and alkyl phenols ethoxylated with 1 to 6 mol ethylene oxideare particularly suitable. Sulfated fatty acid alkanolamides andsulfated fatty acid monoglycendes are also suitable.

[0061] The sulfonate-type surfactants are primarily the alkylbenzenesulfonates containing C₉₋₁₅ alkyl groups, sulfosuccinic acid monoestersand diesters containing 6 to 22 carbon atoms in the alcohol componentsand the esters of a-sulfofatty acids, for example the a-sulonated methylor ethyl esters of hydrogenated coconut oil, palm kernel oil or tallowfatty acids. Other suitable surfactants of the sulfonate type are thealkane sulfonates obtainable from C₁₂₋₁₈ alkanes by sulfochlorination orsulfoxidation and subsequent hydrolysis or neutralization or by additionof bisulfite onto olefins and also olefin sulfonates, i.e. mixtures ofalkene and hydroxyalkane sulfonates and also disulfonates which areobtained, for example, from long-chain monoolefins with a terminal orinternal double bond by sulfonation wit gaseous sulfur trioxide andsubsequent alkaline or acidic hydrolysis of the sulfonation products.

[0062] Bleaching agents are preferably selected from the type containingperoxygen, as hydrogen peroxide, alkali perborate, alkali percarbonate,alkali persilicate and/or alkali persulfate. Particularly preferred aresodium perborate monohydrate and sodium percarbonate. Bleaching agentsmay be present in amounts of 5% by weight to 25% by weight, moreparticularly 7% by weight to 20% by weight.

[0063] Bleach activator compounds include in particular N- or O-acylcompounds, for example polyacylated alkylene diamines, more particularlytetraacetyl ethylene diamine, N-acylated triazines, more particularly1,5-diacetyl2,4-dioxohexahydro-1,3,5-triazine, acylated glycolurils,more particularly tetraacetyl glycoluril, N-acylated hydantoins,hydrides, triazoles, urazoles, diketopiperazines, sulfuryl amides andcyanurates, also carboxylic anhydrides, more particularly phthalicanhydride, carboxylic acid esters, more particularly sodiumisononanoyloxy benzene sulfonate, and acylated sugar derivatives, moreparticularly pentaacetyl glucose. The bleach activator may be coated inthe usual way with shell-forming substances or may be granulated,optionally using granulation aids, and if desired may contain otheradditives, for example dye. A bleach activator which formsperoxocarboxylic acids with 2 to 12 carbon atoms, in particularperoxoacetic acid, under the washing conditions is preferably used. Aparticularly preferred bleach activator is tetraacetyl ethylene diamine(TAED) granulated with carboxymethyl cellulose with average particlesizes of 0.01 mm to 0.8 mm, which may be produced by the processdescribed in European Patent EP-B-0 037 026. In addition to the abovementioned bleach activators or even subtituting them so-called bleachcatalysts may be used, which are transition metal complexes, for exampleas described in

[0064] Enzymes which may be present in the detergents according to theinvention, in addition to the cellulase according to the definition, areproteases, lipases, cutinases, amylases, pullulanases, other cellulases,hemicellulases, xylanases, oxidases and/or peroxidases. They may bepresent in amounts up to 5% by weight, preferably 0.2% by weight to 2%by weight.

[0065] The detergent compositions of the invention may be formulated inany convenient form e.g. as a powder or liquid. For the production ofdetergents with high apparent density of e.g. 650 g/l to 950 g/l amethod using an extrusion step, as described in European patent EP-B-0486 592, is preferred.

[0066] Fabric softening compositions comprising the inventive cellulasemay comprise further to this cellulose cationic surfactants, preferablyof the so-called esterquat type, which are capable of fabric softeningand which may increase the fabric softening properties of thecompositions.

EXAMPLES Example 1 Production of Cellulases Screening for CellulaseProducing Microorganisms

[0067] Two methods were applied for the isolation of cellulase-producingmicroorganisms:

[0068] 1) the soil and water samples were suspended in 0.85% salinesolution and directly used in the carboxymethyl cellulose (CMC)-agardiffusion assay for detection of cellulase producing colonies.

[0069] 2) The soil and water samples were enriched for cellulasecontaining strains by incubation in a cellulose containing liquidminimal medium or GAM-medium for 1 to 3 days at 40° C. Cultures thatshowed bacterial growth were analyzed for cellulase activity using theCMC-agar diffusion assay for detection of cellulase producing colonies.

Isolation of Alkalitolerant, Cellulase Producing Strains

[0070] Strains that showed clearing zones in the agar diffusion assaywere fermented in 25 milliliter GAM-medium in 100 milliliter shakeflasks in an Incubator Shaker (New Brunswick Scientific, Edison, N.J.,USA), at 250 r.p.m. at 40° C. for 72 hours. CMCase activity wasdetermined in the culture broth at pH 9 and 40° C.

Isolation of Cellulase Genes

[0071] Genomic gene libraries of the alkalitolerant cellulase producingstrains were constructed in plasmid pTZ18R (Mead, D. A., et al. (1986)Protein Engineering 1, 67). Recombinant clones were screened by agardiffusion on CMC-agar as described by Wood, P. J., et al. 188) Methodsin Enzymology 160, 59-74. Strains that showed clearing zones around thecolony were isolated. The CMCase activity of the recombinant strains wasdetermined after fermentation for 48 hours at 300° C. in 4*YEP-medium.The plasmid DNA of the recombinant strains was isolated and the insertswere characterized by restriction enzyme analysis and nucleotidesequence analysis.

Media

[0072] The minimal medium (pH 9.7) used in the CMC-agar diffusion assayand the enrichment procedure, consisted of KNO₃ 1%, Yeast extract(Difco) 0.1%, KH₂PO₄ 0.1%, MgSO₄.7H₂O 0.02%, Na₂CO₃ 1%, NaCl 4% and0.25% CMC (Sigma C4888). For solidification 1.5% agar was added.

[0073] The complex medium (GAM) used for enzyme production of the donorstrains consisted of Peptone (Difco) 0.5%, Yeast extract (Difco) 0.5%,Glucose.H₂O 1%, KH₂PO₄ 0.1%, MgSO₄.7H₂O 0.02%, Na₂CO₃ 1%, NaCl 4%. ThepH was adjusted to 9.5 with 4M HCl after which 1% CMC was added.

[0074] The complex medium (4*YEP) used for the enzyme production in E.coli recombinant strains consisted of Yeast extract (Difco) 4%, Peptone(Difco) 8%, lactose 0.2%, 100 μg/ml ampicilline.

CMC-agar Diffusion Assay for Colonies

[0075] Cell suspensions in 0.85% saline solution were plated onCMC-containing minimal medium. After incubation for 1 to 3 days at 400°C., the plates were replica plated and the parent plate was flooded with0.1% Congo Red for 15 minutes. The plates were destained with 1M NaClfor 30 minutes. The strains that showed a clearing zone aroung thecolony were isolated as potential cellulases producing microorganisms.

CMC-agar Diffusion Assay for Liquid Fractions

[0076] Aliquots of 40 μl of enzyme solution or fermentation broth werepipetted in wells punched out from a layer of 5 mm of minimal medium ina petri dish. After incubation for 16 hours at 40° C. cellulase activitywas detected by Congo Red/NaCl treatment. The diameter of the clearingzone is a measure for the CMCase activity.

Resulting Cellulase

[0077] These experiments resulted in the isolation of a cellulaseproducing microorganism which was deposited thereafter as CBS 670.93.The microorganism was classified as a new species of the genus Bacillus.Cloning experiments with the CBS 670.93 strain as a donor strainresulted in the isolation of an E. coli clone which was able to producea cellulase called BCE 103. The nucleotide sequence of the gene codingfor said cellulase was analysed. From the cellulase BCE 103 theN-terminal amino acid sequence was determined using standard methods forobtaining and sequencing peptides (Finlay & Geisow (Eds.), ProteinSequencing—a practical approach, 1989, IRL Press). The amino acidsequence of the cellulase was deduced from the nucleotide sequence,using the N-terminal amino acid sequence for the starting point of themature protein.

[0078] The nucleotide sequence for BCE 103 is shown in SEQ ID No.1 andthe amino acid sequence is shown in SEQ ID No.2.

Purification of the Cellulase

[0079] After the fermentation the cells were separated from the cultureliquid by centrifugation (8000 rpm). The cellulase in the supernatantwas precipitated with ammonium sulphate (65% saturation) The precipitatewas dissolved in 25 mM phosphate buffer pH 7+5 mM EDTA until aconductivity of 7 mS/cm. This solution was applied to a Q-Sepharose FF(diameter 5 cm, length 10 cm) Anion Exchange column, after which thecolumn was washed with 25 mM phosphate buffer pH 7+5 mM EDTA until anabsorbency of 0.2 AU. A gradient of 0 to 0.5 M NaCl in 25 mM phosphatepH 7 was applied to the column in 80 minutes followed by a gradient from0.5 to 1 M NaCl in 10 minutes. Depending on which cellulase was appliedto the column, elution took place in the first or the second gradient.After elution the column was cleaned (uprow) with 1 M NaOH andequilibrated again with 25 mM phosphate pH 7+5 mM EDTA. Depending on theelution the obtained cellulase had a purity of up to about 80%.

Characterization CMCase assay

[0080] Assays for cellulase activity were performed using modifiedmethods of the PAHBAH method (Lever M. Anal. Biochem. 1972, 47, 273-279and Lever M. Anal. Biochem. 1977, 81, 21-27).

Procedure

[0081] A test tube is filled with 250 μl 2.5% CMC in 50 mM glycinebuffer pH 9 (CMC-low viscosity is purchased from Sigma) and 250 μlaliquots cellulase, diluted in the appropriate buffer. The test tube isincubated for 30 minutes at 40° C. in a waterbath, whereafter 1.5 ml ofa daily fresh prepared PAHBAH solution (1% PAHBAH in 100 ml 0.5 M NaOHwith 100 μl bismuth solution (containing 48.5 g bismuth nitrate, 28.2 gpotassium sodium tartrate and 12.0 g NaOH in 100 ml) is added. Themixture is heated at 70° C. for 10 minutes, after which it is cooled onice for 2 minutes. The absorption is measured at 410 nm. To eliminatethe background absorbance of the enzyme samples a control experiment isexecuted as follows: a tube with substrate is incubated under the sameconditions as the test tube. After the incubation 1.5 ml PAHBAH and theenzyme preparation is added (in this order). One unit (U) is defined asthe amount of enzyme producing 1 μmol of glucose from CMC equivalentdetermined as reducing sugars per minute per gram product.

[0082] The buffer used for the determination of the pH/temperatureprofiles is a phosphate/citrate system. The pH/temperature profiles weredetermined using a fixed enzyme concentration which fits in the linearrange of the dose response profile measured at pH 7 and 40° C. Thisenzyme concentration was used for the measurement of the activitiesunder all other determined conditions.

[0083] The results for the cellulase BCE 103 are shown in FIG. 1. Thiscellulase shows good activities at alkaline pH, which makes it suitablefor application in detergents with an alkaline pH.

Example 2

[0084] Similar procedures starting with the alkalophilic bacillus strainCBS 669.93 resulted in cellulase BCE 113. The results for this cellulaseBCE 113 are shown in FIG. 2. This cellulase also shows good activitiesat alkaline pH, which makes it suitable for application in detergentswith an alkaline pH.

Example 3. Measurement of Tensile Strength and Antipilling

[0085] As described for the evaluation of TSL, washing experiments wereperformed using as detergent matrix a Colour Detergent without bleach,without perfume and enzymes (105 g detergent per wash cycle, pH 10.5),as washing machine a type Miele® W 717, temperature 40° C., program“Normalprogramm”, with water of a hardness of 16°dH (German hardness),wash load 3.5 kg, 25 washes.

[0086] Experiments using a composition according to the invention (D1)as well as comparisons (C1 to C3) were run in parallel in identicalmachines:

[0087] C1: detergent matrix without cellulose

[0088] C2: detergent matrix+0.288 mg endoglucanase V from Humicolainsolens

[0089] C3: detergent matrix+cellulase mixture from Humicola insolenssold as granules Celluzyme® 0.7T

[0090] D1: detergent matrix+0.288 mg cellulase BCE 103

[0091] D2: detergent matrix+0.288 mg cellulase BCE 113 TABLE 1 Resultsof TSL-measurements [%] Composition TSL C1  0 C2 100 C3  38 D1  12

[0092] Using washing machines of type Miele® W 914, under otherwiseidentical conditions, gave the following results: TABLE 2 Results ofTSL-measurements [%] Composition TSL C1 0 C2 100 D2 0.6

Example 3 Measurement of Antipilling and Calculation of the Ratio TSL toAP

[0093] The evaluation of antipilling properties was done with increasedconcentrations of cellulases for better quantitative evaluation of theeffect. A Colour Detergent (5 g/l, to wash cycles at 40° C.) with theaddition of cellulase as given in Table 3 was used on “pilled” sweatshirt cotton material (washed 25 times at 60° C. with a detergentwithout cellulase). Evaluation of the pilling was done with the opticalmeasurement system as described before; a degree of pilling of 0% wasassigned to the “pilled” material. TABLE 3 Results of AP-measurements[%] degree of pilling Enzyme concentration EG V BCE 103 AP [%] of BCE103 25 μg/ml −12.8% −8.4% 65% 37.5 μg/ml −16.0% −9.6% 59% 50 μg/ml−22.8% −15.6% 68%

[0094] An average AP of 64% can be calculated for BCE 103 cellulase. BCE113 cellulase showed under the same conditions an average AP of 100%.

[0095] Using the values for TSL in Tables 1 and 2, the ratios of TSL toAP for the various cellulases are as in the following Table 4: TABLE 4Ratio TSL to AP Enzyme Ratio EG V 1 BCE 103 ≈0.2 BCE 113 ≈0.02

Example 4 Further Test Procedures Anti Redeposition Test

[0096] 20 ml 0.5% pigmented soil (fresh prepared, daily and consistingof 86% kaolin, 8% soot (Flammruβ 101, obtained from Degussa AG), 4% ironoxide black and 2% iron oxide yellow (from Henkel Genthin GmbH)), in adetergent (Persil color® without enzymes, 5 g/l, pH 8.5) was, underagitating (90 rpm) incubated with white cotton fabric (prewashed, 5 cmdiameter, obtained from Windelbleiche, Krefeld). Cellulase was addeduntil a final concentration of 1 mU/ml. The mixture was incubated for 30minutes at 40° C., 90 rpm. As a control the same incubation was carriedout without the addition of cellulase. After the incubation the fabricwas rinsed thoroughly with running cold water. After drying thewhiteness of the fabric was measured by remission (4 measurements perfabric) using a Micro color Dr. Lange® Colourimeter. The control valuewas substracted from the sample value. The results, expressed as deltaRem, are shown in Table 5.

Fiber Damage Test

[0097] One pad of cotton wool (100% cotton, Warenhandels GmbH, Buchholz,Marko Olivia, Selling agency: Aldi) was incubated in 40 ml wash liquor(Persil color®) without enzyme, 5 g/l pH 8.5), cellulose at a finalconcentration of 1 mU/ml was added in a sealed flask and incubated for20 hours at 40° C. under agitation (90 rpm). After the incubation, fiberdamage was monitored by the measurement of the quantity of the reducingsugars in solution, using the PAHBAH method described in Example 1. As acontrol the same incubation was carried out without the addition ofcellulase. The results are shown in Table 5.

Adsorption Test

[0098] White cotton fabric (Windelbleiche, Bielefeld) prewashed withgerman Persil® without enzymes at 60° C., was out round to 9 cm diameter(approx. 0.920 gram). One cotton swatch was incubated in 50 ml 50 mMglycine-NaOH buffer pH 9 including 0.1% SDS and 1 ml cellulase sample(600 mU/ml) for 60 minutes at 300° C. 2 ml samples were taken at T=0 andat T=60 minutes and were diluted directly (1:2) with 50 mM MES buffer pH6.5 and stored at 4° C. until measurement. As control the sameincubation was carried out without the addition of cotton textile. Theactivity measurement was determined with a PAHBAH method as described inExample 3, but at pH 6.5 in 50 mM MES buffer. The adsorption wasexpressed as relative adsorption where the activity applied at the startof the experiment was set as 100%, T=0. 100% activity value—remainingactivity (%)=adsorption (%). The results are shown in Table 5. TABLE 5Results of the Antiredeposition Test, Fibre Damage Test and AdsorptionTest Antiredeposition Fibre Damage Enzyme [delta REM] [mU] Adsorption[%] BCE 103 5.0 0.025  7 KAC ®^(a)) 7.5 0.006  0 EG V 1.2 0.155 36

Softening Test

[0099] The softness of fabrics treated as in Example 3, but after 15wash cycles, was rated by an expert panel (5 persons) who awarded gradesbetween 0 (fabric washed 25 times with a detergent without cellulase)and 6 (fabric prior to any wash) by the feel of the fabrics.Compositions as defined in Example 2 were used in the washings. Theaverage rates are given in Table 6 it can be seen that the compositionsaccording to the invention showed the best performance. TABLE 6 Resultsof the Softening Test Composition Rate C1 0 C2 2.1 C3 1.5 D1 2.3 D2 2.2

LEGEND TO THE FIGURES

[0100]FIG. 1 shows the relative activities of the cellulase BCE 103. InExample 1 this figure is referred to as the pH/temperature profiles. Allactivities for both 40 and 60° C. are related to the highest activitywhich is fixed on 100%.

[0101]FIG. 2 shows the relative activities of the cellulase BCE 113.

[0102]FIG. 3 shows the DNA sequence (SEQ ID No.1) and deduced amino acidsequence (SEQ ID No.2) of the 50 kD cellulase derived from CBS 670.93with the leader peptide sequence shaded, which upon secretion is cleavedto yield the mature enzyme.

[0103]FIG. 4 shows the DNA sequence (SEQ ID No.4) and deduced amino acidsequence (SEQ ID No.3) of the 63 kD cellulase derived from CBS 669.93with the leader peptide sequence underlined, which upon secretion iscleaved to yield the mature enzyme.

1 2 1 467 PRT Bacillus sp. CBS 670.93 1 Met Lys Lys Ile Thr Thr Ile PheAla Val Leu Leu Met Thr Leu Ala 1 5 10 15 Leu Phe Ser Ile Gly Asn ThrThr Ala Ala Asp Asp Tyr Ser Val Val 20 25 30 Glu Glu His Gly Gln Leu SerIle Ser Asn Gly Glu Leu Val Asn Glu 35 40 45 Arg Gly Glu Gln Val Gln LeuLys Gly Met Ser Ser His Gly Leu Gln 50 55 60 Trp Tyr Gly Gln Phe Val AsnTyr Glu Ser Met Lys Trp Leu Arg Asp 65 70 75 80 Asp Trp Gly Ile Thr ValPhe Arg Ala Ala Met Tyr Thr Ser Ser Gly 85 90 95 Gly Tyr Ile Asp Asp ProSer Val Lys Glu Lys Val Lys Glu Thr Val 100 105 110 Glu Ala Ala Ile AspLeu Gly Ile Tyr Val Ile Ile Asp Trp His Ile 115 120 125 Leu Ser Asp AsnAsp Pro Asn Ile Tyr Lys Glu Glu Ala Lys Asp Phe 130 135 140 Phe Asp GluMet Ser Glu Leu Tyr Gly Asp Tyr Pro Asn Val Ile Tyr 145 150 155 160 GluIle Ala Asn Glu Pro Asn Gly Ser Asp Val Thr Trp Asp Asn Gln 165 170 175Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro Val Ile Arg Asp Asn Asp 180 185190 Pro Asn Asn Ile Val Ile Val Gly Thr Gly Thr Trp Ser Gln Asp Val 195200 205 His His Ala Ala Asp Asn Gln Leu Ala Asp Pro Asn Val Met Tyr Ala210 215 220 Phe His Phe Tyr Ala Gly Thr His Gly Gln Asn Leu Arg Asp GlnVal 225 230 235 240 Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile Phe Val SerGlu Trp Gly 245 250 255 Thr Ser Ala Ala Thr Gly Asp Gly Gly Val Phe LeuAsp Glu Ala Gln 260 265 270 Val Trp Ile Asp Phe Met Asp Glu Arg Asn LeuSer Trp Ala Asn Trp 275 280 285 Ser Leu Thr His Lys Asp Glu Ser Ser AlaAla Leu Met Pro Gly Ala 290 295 300 Asn Pro Thr Gly Gly Trp Thr Glu AlaGlu Leu Ser Pro Ser Gly Thr 305 310 315 320 Phe Val Arg Glu Lys Ile ArgGlu Ser Ala Ser Ile Pro Pro Ser Asp 325 330 335 Pro Thr Pro Pro Ser AspPro Gly Glu Pro Asp Pro Gly Glu Pro Asp 340 345 350 Pro Thr Pro Pro SerAsp Pro Gly Glu Tyr Pro Ala Trp Asp Ser Asn 355 360 365 Gln Ile Tyr ThrAsn Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln 370 375 380 Ala Lys TrpTrp Thr Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro 385 390 395 400 TrpGlu Pro Leu Lys Ser Asp Pro Asp Ser Gly Glu Pro Asp Pro Thr 405 410 415Pro Pro Ser Asp Pro Gly Glu Tyr Pro Ala Trp Asp Ser Asn Gln Ile 420 425430 Tyr Thr Asn Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys 435440 445 Trp Trp Thr Gln Asn Gln Glu Pro Gly Asn Pro Tyr Gly Pro Trp Glu450 455 460 Pro Leu Asn 465 2 574 PRT Bacillus sp. 669.93 2 Met Lys TrpMet Lys Ser Met Val Trp Leu Ala Val Val Leu Val Val 1 5 10 15 Ser PheVal Ala Pro Ala Val Ser Ser Ala Asn Glu Asp Val Lys Thr 20 25 30 Leu AspIle Gln Ser Tyr Val Arg Asp Met Gln Pro Gly Trp Asn Leu 35 40 45 Gly AsnThr Phe Asp Ala Val Gly Gln Asp Glu Thr Ala Trp Gly Asn 50 55 60 Pro ArgVal Thr Arg Glu Leu Ile Glu Arg Ile Ala Asp Glu Gly Tyr 65 70 75 80 LysSer Ile Arg Ile Pro Val Thr Trp Glu Asn Arg Ile Gly Gly Ala 85 90 95 ProAsp Tyr Pro Ile Asp Pro Gln Phe Leu Asn Arg Val Asp Glu Val 100 105 110Val Gln Trp Ala Leu Glu Glu Asp Leu Tyr Val Met Ile Asn Leu His 115 120125 His Asp Ser Trp Leu Trp Ile Tyr Glu Met Glu His Asn Tyr Asn Gly 130135 140 Val Met Ala Lys Tyr Arg Ser Leu Trp Glu Gln Leu Ser Asn His Phe145 150 155 160 Lys Asp Tyr Pro Thr Lys Leu Met Phe Glu Ser Val Asn GluPro Lys 165 170 175 Phe Ser Gln Asn Trp Gly Glu Ile Arg Glu Asn His HisAla Leu Leu 180 185 190 Asp Asp Leu Asn Thr Val Phe Phe Glu Ile Val ArgGln Ser Gly Gly 195 200 205 Gln Asn Asp Ile Arg Pro Leu Val Leu Pro ThrMet Glu Thr Ala Thr 210 215 220 Ser Gln Pro Leu Leu Asn Asn Leu Tyr GlnThr Ile Asp Lys Leu Asp 225 230 235 240 Asp Pro Asn Leu Ile Ala Thr ValHis Tyr Tyr Gly Phe Trp Pro Phe 245 250 255 Ser Val Asn Ile Ala Gly TyrThr Arg Phe Glu Glu Asp Ser Lys Arg 260 265 270 Glu Ile Ile Glu Thr PheAsp Arg Val His His Thr Phe Val Ala Arg 275 280 285 Gly Ile Pro Val ValLeu Gly Glu Phe Gly Leu Leu Gly Phe Asp Lys 290 295 300 His Thr Gly ValIle Gln Gln Gly Glu Lys Leu Lys Phe Phe Glu Tyr 305 310 315 320 Leu IleHis His Leu Asn Glu Arg Asp Ile Thr His Met Leu Trp Asp 325 330 335 AsnGly Gln His Phe Asn Arg His Thr Tyr Glu Trp Tyr Asp Glu Glu 340 345 350Leu Phe Asp Met Leu Arg Ala Ser Trp Gly Gly Arg Ser Ser Val Ala 355 360365 Glu Ser Asn Phe Ile Tyr Leu Lys Gln Gly Asp Arg Ile Ala Asp Ala 370375 380 Thr Val Thr Leu Gln Leu His Gly Asn Glu Leu Thr Gly Leu Gln Ala385 390 395 400 Asn Gly Gln Arg Leu Thr Pro Gly Gln Asp Tyr Glu Leu AsnGly Glu 405 410 415 Arg Leu Thr Val Lys Ala His Val Leu Ser Ala Ile AlaGly Ser Gly 420 425 430 Thr Leu Gly Thr Asn Gly Met Val Thr Ala Glu PheAsn Arg Gly Ala 435 440 445 Asp Trp His Phe Arg Val Asn Thr Tyr Arg ThrPro Val Leu Gln Ser 450 455 460 Thr Gln Gly His Val Ser Asn Phe Ser IlePro Ala Ser Phe Asn Gly 465 470 475 480 Asn Ser Leu Ala Thr Met Glu AlaVal Tyr Val Asp Gly Gly Asn Ala 485 490 495 Gly Pro Gln Asp Trp Thr SerPhe Lys Glu Phe Gly Tyr Ala Phe Ser 500 505 510 Pro Ser Tyr Asp Thr HisGlu Ile Lys Leu Thr Glu Ala Phe Phe Arg 515 520 525 Glu Val Arg Asp GlyGlu Val Arg Leu Thr Phe His Phe Trp Ser Gly 530 535 540 Glu Ile Val AsnTyr Thr Ile Ile Lys Asn Gly Asn Gln Val Thr Gly 545 550 555 560 Ile AlaAla Gln Thr Thr Asn Ser Lys Asn Lys Asn Lys Lys 565 570

1. Use of a single cellulase with a ratio of tensile strength loss (TSL)to antipilling properties (AP) below 1 in aqueous laundry solutions. 2.Use according to claim 1, characterized in that the aqueous laundrysolution comprises the cellulase in concentrations of 0.01 mg/l to 0.2mg/l more particularly 0.015 mg/l to 0.1 mg/l.
 3. Use of a singlecellulase with a ratio of TSL to AP below 1 to provide an anti-greyingeffect to fabrics, especially colored fabrics.
 4. Use of a singlecellulase with a ratio of TSL to AP below 1 to provide a softeningeffect to fabrics.
 5. Use of a single cellulase with a ratio of TSL toAP below 1 to provide colour clarification to fabrics or to inhibitcolor deterioration of fabrics, especially colored fabrics.
 6. Use of asingle cellulase with a ratio of TSL to AP below 1 to inhibit thewrinkling of fabrics and to ease the ironing of fabrics.
 7. Useaccording to any of claims 1 to 6, characterized in that the ratio ofTSL to AP is below 0.8 and more particularly in the range of 0.001 to0.5.
 8. Use according to any of claims 1 to 7, characterized in that thecellulase is obtainable from Bacillus sp. CBS 669.93 or CBS 670.93. 9.Use according to any of claims 1 to 8, characterized in that thecellulase has the amino acid sequence as listed in SEQ ID No.2 or aderivative thereof.
 10. Use according to any of claims 1 to 8,characterized in that the cellulase has the amino acid sequence aslisted in SEQ ID No.3 or a derivative thereof.
 11. Use according toclaim 9 or 10, characterized in that the cellulase has an amino acidsequence with greater than 58%, preferably greater than 80% and moreparticularly greater than 90% sequence identity and/or greater than 72%.preferably greater than 80% and more particularly greater than 90%sequence similarity to the amino acid sequence as listed in SEQ ID No.2and/or SEQ ID No.
 3. 12. A detergent composition which comprises asingle cellulose with a ratio of TSL to AP below
 1. 13. A detergentcomposition according to claim 12, characterized in that it comprises0.8 ppm to 80 ppm, more particularly 1 ppm to 40 ppm of the cellulase.